Clinical Significance of TUBGCP4 Expression in Hepatocellular Carcinoma

We aim to investigate the expression and clinical significance of the tubulin gamma complex-associated protein 4 (TUBGCP4) in hepatocellular carcinoma (HCC). The mRNA expression of TUBGCP4 in HCC tissues was analyzed using The Cancer Genome Atlas (TCGA) database. Paired HCC and adjacent nontumor tissues were obtained from HCC patients to measure the protein expression of TUBGCP4 by immunohistochemistry (IHC) and to analyze the relationship between TUBGCP4 protein expression and the clinicopathological characteristics and the prognosis of HCC patients. We found that TUBGCP4 mRNA expression was upregulated in HCC tissues from TCGA database. IHC analysis showed that TUBGCP4 was positively expressed in 61.25% (49/80) of HCC tissues and 77.5% (62/80) of adjacent nontumor tissues. The Chi-square analysis indicated that the positive rate of TUBGCP4 expression between HCC tissues and the adjacent nontumor tissues was statistically different (P < 0.05). Furthermore, we found that TUBGCP4 protein expression was correlated with carbohydrate antigen (CA-199) levels of HCC patients (P < 0.05). Further, survival analysis showed that the overall survival time and tumor-free survival time in the TUBGCP4 positive group were significantly higher than those of the negative group (P < 0.05), indicating that the positive expression of TUBGCP4 was related to a better prognosis of HCC patients. COX model showed that TUBGCP4 was an independent prognostic factor for HCC patients. Our study indicates that TUBGCP4 protein expression is downregulated in HCC tissues and has a relationship with the prognosis of HCC patients.


Introduction
Hepatocellular carcinoma (HCC) is one of the most common and deadly digestive tract tumors, accounting for 90% of primary liver cancer, and is the fourth leading cause of cancerrelated death worldwide [1][2][3]. The low rate of early diagnosis is a major factor leading to a poor overall survival of HCC patients, which limits the effectiveness of treatment [4]. Therefore, we need to have a better understanding of HCC to improve the early diagnosis and treatment of HCC. At present, the complex genome map of HCC has been widely studied to find the correlation between genes and the prognosis of patients [5]. It is of great theoretical significance and clinical application value to search for predictive markers for recurrence and metastasis of HCC and explore potential targets of intervention therapy for improving the prognosis of patients with HCC [6][7][8][9]. The pathogenesis, prognostic markers, molecular targets, and carcinogenesis mechanism of HCC have been extensively studied [10][11][12][13], but the exact molecular mechanism of HCC is still not fully understood.
Tubulin gamma complex-associated protein 4 (TUBGCP4) is a part of the γ-tubulin ring complex, which is essential for the microtubule nucleation of the centrosome. In particular, it plays a crucial role in the formation of cilium structure, so the assembly or function defects of cilia may cause syndromic cilia lesions [14]. Under normal physiological conditions, TUBGCP4 functions in embryonic development and retinal dynamic balance and has a gene dosage effect [15][16][17]. The abnormal expression of TUBGCP4 can lead to many diseases, especially malignant tumors. It has been found that TUBGCP4 protein is abnormally expressed in ovarian epithelial carcinoma [18], mantle cell lymphoma, and myeloma [19] and is closely related to tumor cell growth and metastasis. However, there are few studies on TUBGCP4 in HCC.
In this study, TUBGCP4 expression was detected in HCC and adjacent nontumor tissues by bioinformatics and immunohistochemistry (IHC) methods. We aimed to analyze the relationship between TUBGCP4 and clinicopathological characteristics as well as HCC prognosis so as to provide a theoretical basis for the clinical application of TUBGCP4 in HCC diagnosis.

Materials and Methods
2.1. Tissue Samples. Eighty paired HCC and adjacent nontumor tissues were collected from HCC patients that had not received any type of treatment before surgery in the First Affiliated Hospital of Guilin Medical University. All patients had complete clinical data that are shown in Table 1 and signed the written informed consent. This study was approved by the Ethics Committee of Guilin Medical University (GLMC2014003) in accordance with the Declaration of Helsinki in 1975.
2.2. Immunohistochemistry (IHC). TUBGCP4 protein was detected by IHC method according to the previous study [20]. To assess the expression of TUBGCP4 in tissues, five different visual fields of view were randomly selected at 400× under microscopy for analysis. The total number of cells and the number of positive cells were counted to score according to the percentage of positive cells. The scores for the percentage of positively stained cells were 0 for ≤5%, 1 for 6-25%, 2 for 26-50%, 3 for 51-75%, and 4 for >75%, respectively. According to the intensity of staining, the scores of unstained, light brown, medium brown, and dark brown were 0, 1, 2, and 3, respectively. Finally, the products of the percentage of positively stained cells and staining intensity scores were used as the final TUBGCP4 staining score: 0 was (-), 1-4 was (+), 5-8 was (++), and 9-12 was (+++). The positive and negative expressions of TUBGCP4 were defined as scores >4 and ≤4, respectively.

Bioinformatic Analysis.
The HCC gene expression profile files were downloaded from The Cancer Genome Atlas (TCGA) database, including 374 samples of HCC and 50 samples of normal liver tissues, and then the data were collated by R language package, and the differences were analyzed. We downloaded RNA sequence data of HCC  Analytical Cellular Pathology patients and analyzed them with Gene Set Enrichment Analysis (GSEA) v4.1.0 software. The significant genes were defined as P < 0:05 and FDR < 0:25. At the same time, to understand the potential function of TUBGCP4, Metascape was used for Gene Ontology (GO) of target genes. Subsequently, we explored the proteins that interact with TUBGCP4 on https://string-db.org/cgi/input.pl and visualized them in the PPI network by Cytoscape v3.7.2 software.

Statistical Analysis.
All the data were statistically processed by SPSS19.0, and the expression difference between HCC and adjacent nontumor tissues was compared by the Chi-square test. The survival probability was estimated by the Kaplan-Meier method, and the survival curve between different groups was analyzed by the Log-rank test. The independent prognostic factors were analyzed by COX proportional hazard model. P < 0:05 was defined as statistically significant.

Expression of TUBGCP4 in HCC Tissues.
We first detected the expression of TUBGCP4 in 80 paired HCC and adjacent nontumor tissues by IHC assay. The positive expression of TUBGCP4 in HCC and adjacent nontumor tissues was 61.25% (49/80) and 77.5% (62/80), respectively. TUBGCP4 protein was mainly located in the cytoplasm (Figure 1(a)). As shown in Table 2, TUBGCP4 protein had a higher expression in the adjacent nontumor tissues than HCC tissues (χ 2 = 7:475, P = 0:006). We then confirmed the mRNA expression of TUBGCP4 in HCC and normal liver tissues from the TCGA database and found that TUBGCP4 mRNA expression in HCC tissues was signifi-cantly higher than that in normal liver tissues (P < 0:05, Figure 1(b)).   3 Analytical Cellular Pathology 1002.375). Further, Log-rank test showed that the overall survival rate of HCC patients in the TUBGCP4 positive group was significantly higher than that in the negative group (P = 0:032, Figure 2(a)). We also found that the postoperative tumor-free survival rate of HCC patients in the TUBGCP4 positive group was significantly higher than that in the negative group (P = 0:003, Figure 2(b)).

Relationship between TUBGCP4 Expression and
In addition, we conducted a COX proportional hazard analysis and found that TUBGCP4 was an independent prognostic factor for HCC patients (P = 0:040), with an OR value of 0.551 (95% CI: 0.312-0.973, Table 3).

The Main Enrichment Pathways of TUBGCP4 in HCC
Tissues. In order to clarify the biological functions of TUBGCP4, the effect of TUBGCP4 on the KEGG pathway was analyzed by GSEA. According to the GSEA consequences of TUBGCP4, 44 significantly downregulated enrichment pathways were screened, including "NOTCH signaling pathway," "WNT signaling pathway," and "MAPK signaling pathway" (Figure 3(a)). Since, they are the three important tumor-related pathways, which supports that TUBGCP4 might be involved in the occurrence and development of HCC. In addition, GO analysis showed that TUBGCP4 was enriched in biological processes of HCC, such as "covalent chromatin modification," "transferase complex," and "mitochondrial protein complex" (Figure 3(b)).
We also constructed a possible protein-protein interaction (PPI) network to study the mechanism of TUBGCP4 in HCC and concluded that TUBGCP4 was closely related to the expression of nonmetastatic cells 7 (NME7), polo-like kinase 4 (PLK4), and other proteins in HCC (Figure 3(c)).

Discussion
Genetic instability is a common feature of malignant tumors, and centrosome abnormalities are related to the induction of aneuploidy in various human malignant tumors [19,21,22]. In structural centrosome proteins, TUBGCP4 encodes γtubulin complex-associated protein 4, which is a template for closely related α and β tubulin microtubule polymerization, and its expression is associated with centrosome aberrations [23]. In recent years, some studies have shown that TUBGCP4 is overexpressed in various malignant tumors and considered to be an oncogene. But the expression of TUBGCP4 might be decreased due to abnormal (exclude exon 16) and unstable mRNA production. However, the production of normal mRNA containing exon 16 is not entirely nonexistent and is also produced by the same allele. According to the American Society of Medical Genetics and Genomics [24], this meaningless mutation can be classified as a possible pathogenic mutation because it is a predicted zero mutation in the TUBGCP4 gene, in which loss of function is a known mechanism of microcephaly and chorioretinopathy type 3 (MCCRP3) [25]. Therefore, as a component of the γ-tubulin ring complex, the analysis of the expression of TUBGCP4 may be helpful to analyze the centrosome aberration of malignant tumors [26], and they are of great significance to the prognosis of various malignant tumors.
Chromosomal number changes occur in almost all types of human malignancy, including the acquisition and loss of entire chromosomes [22]. Therefore, the regulation mechanism of fidelity of mitotic chromosome separation in mammalian cells is a hot topic in tumor biology research. Importantly, Kramer et al. found that the abnormality of the centrosome is related to the occurrence of aneuploidy in different solid tumors caused by poor chromosome segregation in the anaphase of mitosis [21]. A previous study has reported that TUBGCP4 mutations existed in epithelial ovarian cancer, and TUBGCP4 involved in tubulin processing was essential for cell growth [18]. In the study of mantle    Analytical Cellular Pathology cell lymphoma, TUBGCP4 expression was found to be associated with both tetraploidization and high levels of centrosome aberrations [19]. For patients with negative 18F-FDGPET/CT tumor localization but positive TUBGCP4 expression, the detection of tumor markers may be helpful for the localization of tumor origin [27]. These studies suggested that the abnormal expression of the TUBGCP4 gene may lead to abnormal centrosome, which may induce aneuploidy of human malignant tumors and then lead to malignant transformation of cells [26,28].
In this study, we found that the mRNA expression of TUBGCP4 in HCC tissues was higher than that in normal liver tissues by bioinformatic analysis, while IHC results showed that the protein expression of TUBGCP4 in HCC tissues was lower than that in adjacent nontumor tissues. Such a result may be due to the different expression of TUBGCP4 caused by posttranslational modification, or it may be due to the small sample size in our study. When we analyzed the relationship between TUBGCP4 expression and the clinicopathological characteristics of HCC patients, we found that TUBGCP4 expression was correlated with CA-199 levels. CA-199, a carbohydrate antigen secreted by digestive tract tumor cell lines, is very low in normal adults (<37 U/mL). When malignant lesions occur in the body, the content of CA-199 in tumor tissues is greatly increased. So its content has become one of the main specific indicators for clinical diagnosis of HCC [29]. Our results imply that TUBGCP4 can be used as a diagnostic marker of HCC through the interaction with CA-199.
GSEA results showed that TUBGCP4 regulated hepatocytes through the MAPK, NOTCH, and WNT signaling pathway. They are the three key cascades regulating cancer progression and have been reported to be important in metastasis of many tumors including HCC, non-small-cell lung cancer and colorectal cancer [30][31][32][33][34]. Further, GO analysis indicated that TUBGCP4 was enriched in biological processes of HCC, such as "covalent chromatin modification," "transferase complex," and "mitochondrial protein complex". These regulatory processes are regulated by a variety of enzymatic proteins whose mutation or abnormal expression can easily lead to a variety of diseases [35], including HCC. Subsequently, to further explore the mechanism of TUBGCP4, we constructed a hypothetical protein network and found that TUBGCP4 plays a role in promoting cancer cell growth and proliferation.
Moreover, we found that the total postoperative survival time and tumor-free survival time in the TUBGCP4 positive group were significantly longer than that of the TUBGCP4 negative group. Further COX analysis showed that TUBGCP4 was an independent prognostic factor for HCC patients. However, whether TUBGCP4 can be used as a potential prognosis marker of HCC still needs to be further verified by prospective, multicenter, and large sample experimental methods.

Conclusion
The downregulation of TUBGCP4 expression is correlated with the prognosis of HCC patients. TUBGCP4 is expected to become a new molecular marker for predicting the prognosis of HCC patients in the future.

Data Availability
The datasets used and analyzed during the current study are available from the corresponding authors on reasonable request.

Ethical Approval
This study was approved by the Ethics Committee of Guilin Medical University, and the written informed consent of each patient participating in the study was obtained.

Conflicts of Interest
The authors declare no conflict of interest.